Association of Cytokine IL-17, IL-4, IL-6, and IL-12 Gene Polymorphisms in Rheumatoid Arthritis Patients in a Tertiary Care Hospital in Bangladesh

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that involves cytokines in its pathogenesis. This study is aimed at investigating if gene polymorphisms in cytokines like IL-17, IL-4, IL-6, and IL-12 affect RA susceptibility and severity in the Bangladeshi population. This was a cross-sectional comparative study that included 40 diagnosed RA patients according to the American College of Rheumatology (ACR) criteria 2010, who were free from other rheumatological diseases, and 40 healthy subjects for comparison. The study used PCR-RFLP to determine the IL-17, IL-4, IL-6, and IL-12 cytokine gene polymorphisms. Patients had a mean age of 37.22 ± 6.70 years. Among the patients, 31 were female and 9 were male. The mean disease duration was 18.11 ± 7.39 months. The study found that rheumatoid arthritis patients with the IL-17F (7488 A/G) polymorphism with GG genotype (P = 0.006, OR = 8.56, 95% CI = 1.77 − 41.33) and IL-12B (1188 A/C) polymorphism with AC (P = 0.012, OR = 3.69, 95% CI = 1.43 − 9.53) and CC (P = 0.013, OR = 7.58, 95% CI = 1.56 − 36.88) genotypes were significantly associated with disease risk. Furthermore, patients with the IL-17F (7488) GG genotype and IL-12B (1188) AC and CC genotypes had higher rheumatoid arthritis disease severity and activity parameters. The study found no significant association between polymorphisms involving IL-4 (590 C/T) and IL-6 (174 G/C) genes and rheumatoid arthritis disease risk in the Bangladeshi population. Gene polymorphisms in cytokines IL-17F (7488 A/G) and IL-12B (1188 A/C) can predict disease susceptibility and severity in Bangladeshi rheumatoid arthritis patients.


Background
Rheumatoid arthritis (RA) is a common systemic inflammatory autoimmune disease that causes synovial inflammation and can result in chronic and irreparable joint degeneration.Multiple factors, including genetic, environmental, and hormonal factors, contribute to the development of certain autoimmune processes [1].According to the global prevalence of RA, Australia, North America, and Western Europe (0.4% to 0.5%) have a higher prevalence of RA than Asia, the Middle East, and North Africa (0.16%) [2].In Bangladesh, the prevalence of rheumatoid arthritis is 1.6%, with women having a much greater frequency (2.4%) than men (0.7%) [3].Genetic predisposition is associated with a 50-60% risk of RA [4].Among non-HLA genetic factors, gene polymor-phisms, particularly cytokines and their receptors, have an essential role in RA pathogenesis [5].
IL-17 is a proinflammatory cytokine that is made up of six ligands and five receptors [6].Among the six ligands, IL-17A and IL-17F have key functional and biological features, and the genes encoding these two cytokines are found on human chromosome 6p12.2[7,8].Chabaud et al. first suggested the role of IL-17 in RA development by demonstrating the presence of this cytokine in the synovial fluid of RA patients [9].IL-17 participates in tissue inflammation and degradation by promoting the production of metallo matrix proteases and other proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), IL-1, and IL-6, which attract neutrophils, macrophages, and lymphocytes to the synovium [10].There is a histidine-to-arginine substi-tution at amino acid 161 in the 3rd exon of the IL-17F gene in the IL-17F 7488 A/G polymorphism [11].The IL-17F (7488 A/G) polymorphism has been shown to be associated with RA in some previous studies [12][13][14].
The cytokine IL-4 modulates a wide range of immunological processes, including immunoglobulin isotype switching, class-II MHC expression by B cells, and the differentiation of certain T-cell subsets [15].C-to-T base replacement is represented by the IL-4-590 promoter polymorphism, which is located at 589 base pairs (bp) before the transcriptional site [16].The IL-4 gene polymorphism (-590 T/C) has been linked to an increased risk of RA in European and Chinese populations [17,18].
Many RA patients have high levels of IL-6 in their blood and synovial fluid.IL-6 induces inflammation and joint degeneration by acting on neutrophils, which produce reactive oxygen intermediates and proteolytic enzymes [19].Polymorphisms involving the IL-6 (-174 G/C) gene with a G-to-C substitution at position -174 have been linked to an increased risk of RA in the Chinese Han population [18].
IL-12 is a proinflammatory cytokine that increases the production of IFN-γ, which is responsible for the differentiation of naive T cells into Th1 cells.It also increases the cytotoxicity of natural killer cells and cytotoxic T lymphocytes [20].Many RA patients' blood and synovial fluid contain higher levels of IL-12 [21].The IL-12B (+1188 A/C) polymorphism was found to increase susceptibility to RA in the Chinese population [22,23].
Genetic polymorphisms usually differ between ethnic groups.Polymorphisms in the IL-17, IL-4, IL-6, and IL-12 genes may contribute to RA pathogenesis and act as a risk or protective factor for the disease.Polymorphisms involving IL-17, IL-4, IL-6, and IL-12 have been shown to be a risk for RA in some Asian countries, including China and Pakistan.In our country, polymorphisms of these cytokines have not yet been studied in RA patients.
1.1.Study Objective.The aim of this study was to determine if there was a link between IL-17, IL-4, IL-6, and IL-12 gene polymorphisms and rheumatoid arthritis disease susceptibility and severity in the Bangladeshi population, which might help in understanding whether these cytokine polymorphisms act as risk factors for the Bangladeshi rheumatoid arthritis population and may help in future treatment development.
1.2.Research Question.Is there any association of cytokine IL-17, IL-4, IL-6, and IL-12 gene polymorphisms with disease susceptibility and severity in rheumatoid arthritis patients in the Bangladeshi population?

Study Design and Patient
Selection.This study was a crosssectional comparative analysis involving 40 patients diagnosed with rheumatoid arthritis according to the 2010 American College of Rheumatology (ACR) criteria [24] and 40 healthy subjects for comparison.All patients were recruited from the Department of Rheumatology, Bangabandhu Sheikh Mujib Medical University (BSMMU), from January 2023 to March 2023.
The exclusion criteria for patients were as follows: (a) patients under 18 years of age and age more than 60 years, (b) patients with other autoimmune diseases, and (c) patients suffering from major illnesses, such as hepatic failure, renal failure, and cancer.
Age and sex-matched 40 healthy subjects were selected from resident doctors, teachers, and laboratory staffs of the Department of Microbiology and Immunology, BSMMU, without any diagnosed autoimmune or rheumatological disease.

Sample Size.
Sample size for this study was calculated by using the following formula (two-tailed Z-test): where n is the estimated sample size, πo is 66% which is the proportion for group 1 (this is the patient or experimental group [13]), π1 is 92% which is the proportion for group 2 (this is reference or healthy subject's group [13]), π1 − πo is the difference to be detected by the study, u is 1.96 (in 95% confidence interval), and v is 0.84.Therefore, the estimated sample size was The sample size was taken 40, due to available sample.So, the sample size was 40 in each group.

Selection Bias.
Based on the exclusion and inclusion criteria, patients and healthy subjects were selected randomly.A structured data format was prepared to collect necessary and similar data from all the patients to exclude data collection bias.
2.5.Patient Data.Relevant data were taken from the patients, including age, sex, disease duration, and number of tender and swollen joints.Rheumatoid arthritis disease severity was assessed by the Disease Activity Score 28 (DAS28).Functional status was assessed by the Health Assessment Questionnaire (HAQ).Patient laboratory investigations, including erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), rheumatoid factor (RF), and anti-cyclic citrullinated peptide antibodies (anti-CCP) were recorded.A total volume of 25 μl was used for PCR comprising 15 μl of master mix (Emerald Amp MAX PCR Master Mix, Takara Bio, Japan), 1 μl forward primer, 1 μl reverse primer, 3 μl nuclease-free water, and 5 μl extracted DNA.
Thermal cycling parameters for IL-17F (7488A/G) were initial denaturation at 94 °C for 3 min; 35 cycles at 94 °C for 30 sec, 60 °C for 30 sec, and 72 °C for 30 sec; and a final extension at 72 °C for 7 min [12].Initial denaturation at 94 °C for 5 min, followed by 40 cycles at 94 °C for 30 sec, 57 °C for 30 sec, and 72 °C for 35 sec, and a final extension at 72 °C for 10 min were the thermal cycling parameters for IL-4 (590 C/T) [18].Initial denaturation at 95 °C for 5 min,     3 International Journal of Rheumatology followed by 35 cycles at 95 °C for 30 sec, 61 °C for 30 sec, and 72 °C for 30 sec, and a final extension at 72 °C for 10 min were the thermal cycling parameters for IL-6 (174 G/C) [25].Initial denaturation at 95 °C for 15 min, followed by 35 cycles at 94 °C for 45 sec, 54 °C for 60 sec, and 72 °C for 60 sec, and a final extension at 72 °C for 10 min were the thermal cycling parameters for IL-12 (1188 A/C) [26].
The digested product was subjected to electrophoresis in a 2% agarose gel stained with ethidium bromide.After electrophoresis, DNA bands were visualized under UV light (Figures 1-4).A work flow diagram has been shown in Figure 5. Table 1 shows the amplicon sizes before and after digestion.

Statistical Analysis.
The SPSS software package version 27 (Strata Corporation, College Station, Texas) was used to analyze the data.Continuous variables were expressed as mean, standard deviation, and median.Categorical variables were expressed as frequency and percentage.The chi-square (χ 2 ) test was used to assess the significant differences in genotype and allele frequencies between the two research groups.The paired t-test was used to examine the association between polymorphisms and disease activity and severity measures.When the P value was < 0.05, it was considered statistically significant.
All participated patients were on methotrexate.But dose, duration of drugs, and their effect on genotype distribution were not assessed in this study.
Table 3 shows the statistically significant differences in genotype distribution and allele frequency of IL-17F (7488 A/G) and IL-12B (1188 A/C) polymorphisms between rheumatoid arthritis patients and healthy subjects.For the IL-17F (7488A/G) polymorphism, a significant association was found between the homozygous mutant GG According to ACR criteria 2010, diagnosed rheumatoid arthritis patients attended in outpatient department of Rheumatology, BSMMU selected as study subjects and age, sex matched healthy persons were selected as healthy subjects for comparison.
After taking informed written consent and proper data collection 3 ml of peripheral venous blood was collected in EDTA tube.
200 L of blood was used for DNA extraction and extracted DNA was kept at −20°C until work.
Obtained results were compared between patient population and healthy subjects for determination of the association of susceptibility of IL-17, IL-4, IL-6, and IL-12 gene polymorphisms in Rheumatoid Arthritis.
Data analysis and result verified by supervisor.
Gene polymorphisms were detected by PCR-RFLP There were no significant differences in the genotype and allele frequency of the IL-4 (590C/T) and IL-6 (174G/C) gene polymorphisms between RA patients and healthy subjects.None had the homozygous CC genotype for the IL-4 (590C/T) polymorphism, and no subjects had the GC and CC genotype for the IL-6 (174G/C) polymorphism.
Disease activity and severity measures of rheumatoid arthritis were substantially (P < 0 05) higher in the IL-17F (7488 A/G) GG genotype than in the AA genotype (Table 4) and in the IL-12B (1188 A/C) AC and CC genotypes than in the AA genotype (Table 5).

Discussion
Rheumatoid arthritis is a multifactorial disease, and multiple genes may interact and influence disease susceptibility, severity, chronicity, and immune response [29].As genetic factors, genetic polymorphisms in cytokines and their receptors can play roles in RA pathogenesis.The associations of polymorphisms in the cytokine genes IL-17F (7488A/G), IL-4 (590C/T), IL-6 (174G/C), and IL-12B (1188A/C) with RA disease susceptibility and severity were assessed in this study.
This study found that IL-17F (7488A/G) polymorphism with homozygous mutant GG genotype was found to be a significant risk for RA patients (P = 0 006, OR = 8 56, 95% CI = 1 77 − 41 33).Patients with the mutant GG genotype had 8.56 times higher risk of RA than healthy subjects.The mutant G allele frequency was significantly (P = 0 039) higher in RA patients than in healthy subjects.Similar findings were found in a study in Tunisia that showed that GG and AG genotypes were associated with RA risk (P < 0 0001), and the mutant G allele distribution was significantly higher in patients than in controls (P = 0 00002) [13].A Polish study also found discernible differences in the genotype distribution and allele frequency of the IL-17F (7488A/G) polymorphism between RA patients and healthy controls [12].In this study, the RA disease parameters DAS28 (5 16 ± 0 89), HAQ (1 12 ± 0 67), RF (268 3 ± 156 5 IU/ml), and anti-CCP   [12].A study in Brazil found no association between the IL-17F (7488A/G) polymorphism and RA disease parameters [27].In this study, there were substantially more homozygous mutant CC genotypes and heterozygous AC genotypes in RA patients than in healthy subjects for the IL-12B (1188A/C) polymorphism.Patients with the CC genotype had a 7.58 times higher risk of RA development, and patients with the AC genotype had a 3.69 times higher risk of RA.The prevalence of the mutant C allele was significantly higher in RA patients than in healthy subjects.These findings were similar to a study in China, where the genotype frequencies of AC+CC of IL-12B (1188 A/C) significantly varied between RA patients and healthy controls, and the frequency of the C allele was significantly higher in RA patients (P ≤ 0 001) [23].Here, patients with AC and CC genotypes had a significant association with RA severity compared to the AA genotype (P < 0 05).A Chinese study found that the IL-12B (1188A/C) AC/CC genotype was a significant risk factor for RA in RF-positive patients (P = 0 04), but they did not find any significant association of the IL-12B AC/CC genotype with DAS28 and HAQ score [22].
Polymorphisms involving the IL-4 (590 C/T) and IL-6 (174 G/C) genes were not associated with RA risk in this study.A similar finding was found in a study in Poland for the IL-4 (590 C/T) polymorphism [30] and a study in Iraq for the IL-6 (174 G/C) polymorphism [31].They did not find these polymorphisms to be risk factors for RA.A study in China showed that IL-4 (590 C/T) and IL-6 (174 G/C) gene polymorphisms were risk factors for RA [18].

Conclusion
The study concluded that the IL-17F (7488 A/G) polymorphism with GG genotype and G alleles is related to the severity and susceptibility to rheumatoid arthritis.The IL-12B (1188A/C) polymorphism with genotypes CC and AC and C alleles is related to the susceptibility and severity of RA disease.The probability of developing rheumatoid arthritis illness was not correlated with the IL-4 (590 C/T) or IL-6 (174 G/C) gene polymorphisms in this study.

Limitations
This was a single center-based study with a small sample size, so the result may not represent whole RA patients in Bangladesh.Effects of treatment on genotype distribution were not considered in this study; it would be better if considered.

2. 8 .
Expected Outcomes.If cytokine IL-17F (7488 A/G), IL-4 (590 C/T), IL-6 (174 G/C), and IL-12 (1188 A/C) gene polymorphisms became statistically significant in RA patients in comparison to healthy subjects, it might be said that these polymorphisms act as risk factor for RA disease pathogenesis in Bangladeshi population.

Table 4 :
Effect of IL-17F (7488 A/G) polymorphism on RA disease severity and activity.

Table 5 :
Effect of IL-12B (1188 A/C) polymorphism on RA disease severity and activity.
* International Journal of Rheumatology